The present invention refers to a measuring chamber for a flow cytometer to measure the fluorescent and scattered light of individual cells or microscopic particles, comprising: a body with a flat surface, an inlet chamber and an outlet chamber within the body, which open on the flat surface, the chambers being positioned one in front of the other and being connected by a channel in the body, the inlet chamber being equipped with an inlet tube and an injection tube and the outlet chamber being equipped with an outlet tube, a transparent plate placed in front of the flat surface, the plate being moveable from a first position detached from the flat surface to a second position in which the plate closes to seal the cheers and the channel, optical means placed in front of the transparent plate to direct light on the particle contained in the channel and receive fluorescent light emitted from the same particle.
In a flow cytometer, some biological cells or other types of microscopic particles are carried by a laminal flow of a liquid, like water, through the focus of an intense, luminous source. The fluorescent and scattered light emitted from each single cell when it passes through the focus are collected by suitable optical means and are directed onto proper light detectors.
The scattered light, obtained in this way, supplies information on the time and structure of the cell while the fluorescent light is a measure of the contents of the cell which had been previously colored with a fluorescent coloring substance which is bound to it in a particular manner.
To obtain the highest level of excitation possible at the focus and, consequently, the maximum sensitivity, the light is concentrated in a very small focus, usually with a length around 100 micrometers. The cells must follow the same route through the focus in order that they can be analyzed in a reproducable manner. To obtain this, the principle of "hydrodynamic foccussing" is used (P. J. Crossland-Taylor, Nature, Volume 171, Pgs. 37-38, 1953).
Hydrodynamic focussing is obtained in a cone nozzle, filled with water. The water, which is pumped in the nozzle, flows with laminal flow toward an opening at the pointed tip of the nozzle so as to form a lamina jet with a typical velocity of a few meters per second. The sample, that is, the suspension of the cells to be analyzed, is injected in the nozzle through a thin tube which has one of its end openings at or very close to the axis of the nozzle. Since the flow inside the nozzle is laminar, the sample is confined in the central part of the flow, both in the nozzle and the jet. Consequently, it follows a route of the jet that is always reproducible in an exact way. The cells are measured when this jet passes through the excitation focus. The principle of hydrodynamic focussing is used in all of the types of flow cytometers. In some cyometers, the opening of the nozzle connects with the atmospheric air, so that the emitted jet will contact said air. An inconvenience of this type of cytometer is that the user can come into contact with the noxious or infected substances.
In other types of cytometers, the opening connects with a narrow tube, preferably of rectangular section (known as "closed measuring chamber"). A significant inconvenience with this last type is that the thin tube is difficult to clean. However, this has the advantage that the user is protected from exposure to disinfecting and/or toxic agents present in the sample.
There are fundamentally two types of flow cytometers: those which use a laser as a source of light of excitation and those which use a high pressure arc lamp which contains xenon or mercury.
In the cytometers which use laser, the scattering of light can be measured in a direction close to that of the laser beam ("low angle light scattering"). This can also be measured at a right angle relative to the laser beam ("large angle light scattering") whereas the fluorescence is collected at a right angle to the laser beam. Said instruments can use either a nozzle that emits an air jet or in a closed measuring chamber.
The cytometers which use the arc lamps often have a microscope lens with oil immersion for the purpose of concentrating as much exciting light as possible on the flow containing the sample. The fluorescent light is usually collected with the same microscopic lens, which, thus, is called epimodal because it is adapted to collect also the fluorescent light emitted from the sample, while the scattered light of the sample can be highlighted in a dark field in the lower part of the cytometer (documents EP-A-0,229,815, U.S. Pat. No. 4,915,501).
The existence of an open measuring chamber to use with cytometers which use an arc lamp is already known (EP-A-0,026,770). In this measuring chamber, a flow of the liquid is confined in a flat layer of water on the open surface of a microscope cover glass. The flow is observed from the opposite side through a microscope lens with oil immersion.
This type of measuring chamber has a serious inconvenience because it exposes the user to the risk of contamination by toxic and/or infected substances. Moreover, the functioning principle implies a limitation of the raze of sample flow, in some cases thereby limiting the measuring velocity.
On the other hand, this measuring chamber is the only one which allows for a precise measurement of the light scattering in cytometers which use arc lamps.
Other measuring chambers for this type of instruments do not permit an efficient measurement of the light scattering, which represents a serious limitation of their possibility of application (documents U.S. Pat Nos. 4,225,229 and 4,954,715).